Overview of Andor Modular Solutions for Microspectroscopy

• Configuration 1
• Configuration 2
• Configuration 3
• Configuration 4
• Configuration 5
• Configuration 6

Microspectroscopy covers a very wide range of spectroscopy modalities with the common character that the spectroscopic measurement is made on the microscopic scale. There are a number of approaches taken by researchers in configuring systems for such measurements, ranging from the acquisition of a fully integrated ‘push-button’ system that requires the minimum of training for the personnel using the equipment, to in-house purpose-built modular systems where the end-users build and integrate their own system from various individual components. This technical note outlines modular solutions in line with the latter approach for a range of different modalities.

Main Components in Integrating a Microspectroscopy System

The main components required to create an integrated system are illustrated in figure 1. Central to the integration and synchronization of the system components is software control. The main components include a microscope, spectrograph, detector, mechanical parts for coupling the spectrograph with the microscope, optical components for manipulation of the light along the optical paths and feet to match the optical heights between the microscope and spectrograph.

Figure 1: The main components of a modular microspectroscopy system

• Raman Microspectroscopy –(inc. Mapping)
• Confocal Raman Microspectroscopy
• Fluorescence Microspectroscopy
• FRET Microspectroscopy
• Confocal FRET Spectroscopy
• Micro-photoluminescence
• Multiphoton Microspectroscopy
• Spectral Flow Cytometry
• Hyperspectral Imaging
• Scanning Tunnelling Luminescent Spectroscopy
• Optical Coherence Tomography
• Diffuse Scattering Microspectroscopy
• Tip Enhanced Raman Spectroscopy
• Surface Enhanced Raman Spectroscopy
• Intra-operative diagnostics – Cancer diagnosis
• Cell imaging (inc. Live) and diagnostics
• Proteomics
• Genomics
• Ophthalmology – diagnosis of diseases in the eye
• Chemical characterisation of Biomolecules
• Nano-particles, Quantum dots, Quantum wires
• Plasmonics
• Nano- and Micro- Optoelectronic
• Thin film characterisation 
• Analysis of Explosive and Hazardous materials
• Graphene and Semiconductor characterisation
• Microfluidics
• Single Molecule Spectroscopy studies

There is a diversity of areas where microspectroscopy is being applied. Depending on the sample to be analysed there may be one or several spectroscopy modalities used, as listed above. For example Raman spectroscopy is being used to characterise live cells, tissue, biomolecules as well as thin films of various materials such as graphene, carbon nanotubes and microfluidic samples. Optical Coherence Tomography (OCT) is used to investigate the retina of the eye and diagnose skin cancers. Within any given modality there is an ever increasing list of variants of the basic technique.

Examples of Common Experimental Configurations

This technical note outlines some common configurations for coupling the spectroscopy system to various types of microscope – taking into account different spectroscopy modalities. A schematic layout is given for each configuration followed by a brief description. A list of possible (example) parts is then given for constructing such a system. For illustration purposes some specific parts from different suppliers are indicated but one should bear in mind that equivalent parts are available from a range of third-party companies and suppliers.

Here is a High Level Summary of Common Arrangements Encountered

• Configuration 1 - Upright microscope – direct coupling
  • c-mount adapter to turning mirror adapter to elevated spectrograph
  • cage system (or relay optic) from top port to elevated spectrograph
  • cage system with periscope between top port and spectrograph
• Configuration 2 - Inverted microscope – direct coupling
  • c-mount adapter directly to side port
  • cage system between side port and spectrograph
  • relay optic between side port and spectrograph
• Configuration 3 - Inverted microscope – direct coupling
  • single mode fiber (for confocal) from microscope to spectrograph
  • multimode and fiber bundles from microscope to spectrograph
  • pinhole on exit port coupled with multimode fiber to spectrograph

• Configuration 4: Upright (and Inverted) Microscopes with Indirect Optical Fiber Coupling to Spectrograph

• Configuration 5: Upright Microscope with Direct Coupling to the Top Port

• Configuration 6: Upright Microscope with Direct Coupling via a Cage System

Reference Documents

1. Specification sheet for ‘Modular Microspectroscopy Solutions’
2. Specification sheet for ‘Kymera 193i Spectrograph’
3. Technical note on ‘Micro-Manager: Integrating solutions for microspectroscopy’
4. Technical note on ‘Kymera 193i: The high performance compact spectrograph’
5. Some examples of suppliers of general optical components: Thorlabs - www.thorlabs.com, Edmund Optics - www.edmundoptics.eu, LINOS - www.qioptiq.com.
6. Specification sheet for ‘Fiber optical bundles
7. Nikon microscopes – www.nikoninstruments.com, Olympus microscopes – www.olympus-ims.com, Leica microscopes – www.leica-microsystems.com, Zeiss microscopes - www.zeiss.com/microscopy
8. Image courtesy of Dr M Pärs and Dr Ahti Niilisk, Institute of Physics, University of Tartu, Estonia

Configuration 1: Inverted Microscope with Directly Coupled Spectrograph

Figure 2: Schematic and picture of a spectrograph directly coupled to the side port of an inverted microscope.

The schematic shows an excitation light source coupled to the microscope using an optical fiber. However, the excitation may be coupled directly to a port – typically the back port – and may be a broad band source or a laser; the configuration will depend on the spectroscopy modality to be used in the experiment. This example could be used for a fluorescence or micro-luminescence application.

List of Parts Needed for Configuration 1

Configuration 2: Inverted Microscope & Direct Coupling to Spectrograph using Cage System

Figure 3: Schematic for a Raman setup with direct coupling of the spectrograph and microscope using a cage system.

The example illustrated in figure 3 looks at a Raman configuration specifically using a cage system to connect the spectrograph, via relay optics, to the side port of an inverted microscope. A 4f optical arrangement is used for the relay optics. Of course a similar arrangement could be used for a photoluminescence experiment with the notch filter removed from the cage and an appropriate choice of filters within the microscope. There are a large number of variations on this type of configuration but all essentially contain these main components. There is an example in the ‘Modular Solutions for Microspectroscopy’ specification sheet [1] which is similar but is based on having 30 mm diameter relay optics instead. A picture of such a cage arrangement between a microscope and the Kymera 193i is shown in figure 4. A schematic for building this example arrangement is shown in figure 5.

List of Parts Needed for Configuration 2

Figure 4: A picture illustrating the connection of a Kymera 193i to the side port of a microscope using a cage system.

Figure 5: Schematic for the build of the cage system used in this configuration.

Configuration 3: Inverted Microscope with Spectrograph Configured for Confocal Spectroscopy

Figure 6: Schematic of setup for a Confocal Fluorescence Spectroscopy experiment. This depicts an inverted microscope with a cage system attached to its side port. The cage system on the left should be considered in plan-view – with a dichroic mirror being used to direct the laser excitation radiation on to the optical path of the microscope. The returning fluorescent signal passes through the dichroic, is then filtered through a pinhole, and finally is focused into the spectrograph.

This example configuration, schematic layout shown in figure 6, considers a setup for Confocal Fluorescence Spectroscopy based around a cage system, relay optics, a dichroic beam-splitter and a pinhole. In this example two arms are used in the cage system, one to introduce the excitation beam from a laser, and the other to deliver the fluorescent emission signal to the spectrograph. A 4f optical arrangement is used for the relay optics

Of course a similar arrangement could be used for a Raman Confocal Spectroscopy configuration with the use of an appropriate laser excitation, laser filter, and a notch or edge filter. Figure 7 shows a detailed schematic of the specific parts used in this configuration for the cage system.

Figure 7: Schematic showing the details of the specific parts used in building of the cage system for this configuration.

Figure 8: Configuring an upright microscope with optical fiber coupling.

Configuration 4: Upright (and Inverted) Microscopes with Indirect Optical Fiber Coupling to Spectrograph

Configurations which use the indirect coupling of optical fibers to the ports of microscopes are quite popular and relatively easy to configure. However, to ensure the best optimised system in terms of spatial resolution and light collection efficiency can prove challenging. This example demonstrates how an optical fiber can be coupled to the top port of a microscope. The fiber collects the sample signal and delivers it into the spectrograph. In some setups the core diameter of the fiber is used to determine and control the spatial resolution. Some variants in this approach use a pinhole in the optical path at the exit port (or elsewhere in the optical path where appropriate) – with a larger diameter single fiber or fiber bundle collecting the emerging light from the pinhole. The size of the pinhole determines the spatial resolution – with the magnification of the objective being taken into account of course. The pinhole acts as a confocal aperture and with the use of an XY motorised stage can be used to carry out 2D Confocal mapping of the sample.

List of Parts Needed for Configuration 4

Configuration 5: Upright Microscope with Direct Coupling to the Top Port

Figure 9: Examples where the spectrograph is coupled directly to the top port of upright microscopes.

It can be relatively straightforward to connect the spectrograph to the top port of an upright microscope, though this depends on the particular microscope involved and the space available. As illustrated in figure 9 the main component needed is a module with a turning mirror which directs the output signal from the top port into the entrance port of the spectrograph.

The main microscope manufacturers supply dual camera modules for the top port where a mirror at 45° to the main axis can be slid in and out of place depending on which optical path is to be used for the output of the microscope. Examples of such modules for dual port tubes are shown in figure 10 for Nikon (Y-QT) and Olympus (U-DPCAD) upright microscopes. The horizontal output from this module can either be coupled directly to the entrance slit housing of the spectrograph – space permitting – or to the relay optic which delivers the beam to the spectrograph sitting at some distance from the microscope. The spectrograph is supported on a table to match the required height of the output port of the microscope.

Figure 10: Examples of dual camera adapters for upright microscopes – Nikon part Y-QT and Olympus U-DPCAD.

There are a range of other options possible with infinity corrected microscopes for accessing the optical path at an exit port; these are best reviewed by consulting the technical details and manuals of the particular microscope concerned [7].

It is possible to set up a system with individual components with parts from one of the optical component suppliers. Below is an example of components from Thorlabs for attaching a turning mirror to the top port of an upright Nikon Eclipse microscope. Figure 11 illustrates how the parts in this example adaptation are put together.

List of Parts Needed for Configuration 5

Figure 11: Schematic showing the details of the example chosen where specific Thorlabs parts are configured to adopt the top port of a Nikon Eclipse E600 microscope for direct coupling to a spectrograph.

Configuration 6: Upright Microscope with Direct Coupling via a Cage System

A common configuration for microspectroscopy involves the coupling of the spectrograph using a cage system to the top port of an upright microscope. Figure 12 shows a picture of one such configuration used for Raman investigations on thin films [8]. Below in figure 13 is a schematic layout of a similar system for Raman mapping. Figure 14 illustrates how the parts for this example may be assembled.

Figure 12: An example of a configuration with an upright microscope and coupling via a cage system [8].

Figure 13: Schematic layout of system for Raman mapping on the microscopic scale based around an upright microscope. The spectrograph and camera are supported on an elevated table to match the height of the microscope port. A schematic layout showing the assembly of the parts used in this example configuration is shown in figure 14.

List of Parts Needed for Configuration 6

Figure 14: Schematic arrangement showing assembly of parts for configuration 6.